Mapping of post-flowering drought resistance traits in grain sorghum: association between QTLs influencing premature senescence and maturity
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O. R. Crasta W. W. Xu D. T. RosenowJ. Mullet H. T. Nguyen
Mapping of post-owering drought resistance traits in grain sorghum:association between QTLs inuencing premature senescenceand maturity
Received: 10 October 1998 /Accepted: 12 July 1999
Abstract The identification of genetic factors underlyingthe complex responses of plants to drought stress providesa solid basis for improving drought resistance. The stay-green character in sorghum (Sorghum bicolor L.Moench)is a post-flowering drought resistance trait, which makesplants resistant to premature senescence under droughtstress during the grainfilling stage. The objective of thisstudy was to identify quantitative trait loci (QTLs) thatcontrol premature senescence and maturity traits, and toinvestigate their association under post-flowering droughtstress in grain sorghum. A genetic linkage map was de-veloped using a set of recombinant inbred lines (RILs)obtained from the cross B35 Tx430, which were scoredfor 142 restriction fragment length polymorphism(RFLP) markers. The RILs and their parental lines wereevaluated for post-flowering drought resistance and ma-turity in four environments. Simple interval mappingidentified seven stay-greenQTLs and twomaturity QTLs.Three major stay-green QTLs (SGA, SGD and SGG)contributed to 42% of the phenotypic variability (LOD9.0) and four minor QTLs (SGB, SGI.1, SGI.2, and SGJ)significantly contributed to an additional 25% of thephenotypic variability in stay-green ratings. OnematurityQTL (DFB) alone contributed to 40% of the phenotypic
variability (LOD 10.0), while the second QTL (DFG)significantly contributed to an additional 17% of thephenotypic variability (LOD 4.9). Composite intervalmapping confirmed the above results with an additionalanalysis of the QTL Environment interaction. Withheritability estimates of 0.72 for stay-green and 0.90 formaturity, the identified QTLs explained about 90% and63% of genetic variability for stay-green and maturitytraits, respectively. Although stay-green ratings weresignificantly correlated (r 0.22, P 0.05) with matu-rity, six of the seven stay-greenQTLswere independent ofthe QTLs influencing maturity. Similarly, one maturityQTL (DFB) was independent of the stay-green QTLs.One stay-green QTL (SGG), however, mapped in the vi-cinity of a maturity QTL (DFG), and all markers in thevicinity of the independent maturity QTL (DFB) weresignificantly (P 0.1) correlated with stay-green ratings,confounding the phenotyping of stay-green. The molec-ular genetic analysis of the QTLs influencing stay-greenandmaturity, together with the association between thesetwo inversely related traits, provides a basis for furtherstudy of the underlying physiological mechanisms anddemonstrates the possibility of improving drought resis-tance in plants by pyramiding the favorable QTLs.
Key words Sorghum bicolor (L) Drought resistance Quantitative trait loci (QTLs) Trait-based QTLpyramiding
Abiotic stress factors, of which drought and high tem-perature are the major ones, are considered to be themajor cause (71%) of yield reductions in crop plants(Boyer 1982). More than 80% of the sorghum in the USis grown under non-irrigated conditions, where water isthe major limiting factor for yield. Despite the majorresearch emphasis during the last two decades on im-proving drought resistance in sorghum (Rosenow et al.1983), progress in this regard has been slow.
Mol Gen Genet (1999) 262: 579588 Springer-Verlag 1999
Communicated by R. Hagemann
O. R. Crasta W. W. Xu H. T. Nguyen (&)Plant Molecular Genetics Laboratory,Department of Plant and Soil Sciences,Texas Tech University, Lubbock, TX 79409, USAE-mail: firstname.lastname@example.orgTel.:+1-806-742-1622; Fax: +1-806-7420775
O. R. CrastaCuraGen Corporation, New Haven, CT 06511, USA
W. W. Xu D. T. RosenowTexas A and M University Agricultural Researchand Extension Center, Box 219, Lubbock, TX 79401, USA
J. MulletDepartment of Biochemistry and Biophysics,Texas A and M University, College Station, TX 77843, USA
Strategies for crop improvement with respect todrought resistance include the identification and selec-tion of traits that, at least partly, contribute to improvedperformance of the crop under drought conditions. Thistrait-based crop improvement strategy allows selectiveaccumulation of the traits that contribute to droughtresistance for a specific target environment (Blum 1983;Rosenow et al. 1983; Ludlow and Muchow 1990).However, the success of this approach is limited by thediculty experienced in identification of genotypes forseveral traits, due to lack of proper control of the in-tensity and timing of stress. Success is further reduced bythe high cost and the large amount of labor involved inconducting such multi-location experiments.
In grain sorghum, the ability to resist premature se-nescence due to post-flowering drought stress is termedthe stay-green trait (Rosenow et al. 1983). Plants withthe stay-green trait resist premature plant and leaf death,develop grain normally, and resist charcoal rot andlodging when exposed to moisture stress during the latestages of grain development (Rosenow and Clark 1981;Rosenow et al. 1983; Rosenow 1984; Tenkouano et al.1993; Walulu et al. 1994). The stay-green phenomenonhas been extensively studied in plants, motivated byseveral economic incentives (Nooden 1988a; Thomasand Smart 1993; Bleecker and Patterson 1997). Recentstudies have demonstrated that leaf senescence is a ge-netically programmed phenomenon (Oh et al. 1997) andthere is growing interest in studying the molecularmechanisms that underlie this process (Buchanan-Wollaston and Ainsworth 1997; Griths et al. 1997;John et al. 1997; Kleber-Janke and Krupinska 1997;Lers et al. 1998).
Crop plants have been selected for early maturityunder terminal drought stress conditions, which in-creases the probability of encountering favorable mois-ture conditions during the more critical reproductivephase (Ludlow and Muchow 1990). While this pro-grammed completion of the life cycle under conditionsof severe terminal drought stress ensures ecienttranslocation of nutrients to the sink, premature senes-cence aects the assimilatory capacity and the durationof the assimilatory phase, resulting in drastic reductionin grain filling. In this study we have identified theMendelian factors influencing stay-green and early ma-turity, and investigated the phenotypic and genetic as-sociation between these two seemingly inversely relatedtraits. Understanding of the genetic association betweenthese two traits facilitates pyramiding of QTLs for im-provement of drought resistance in crop plants.
Materials and methods
Two genotypes, B35 and Tx430 were selected because they showdistinct dierences in drought response and yield potential. B35 hasoutstanding post-flowering (stay-green) drought resistance. How-ever, it has a relatively low yield potential. Tx430 is a high-yielding
line with exceptionally wide adaptation and is used worldwide inbreeding programs. Tx430, however, is susceptible to post-flower-ing drought stress. The F1 lines obtained from the crossB35 Tx430 were selfed in all successive generations to produceone F6 line from each of the 96 F2 plants. The seeds from each ofthe 96 F6 lines were bulked and used for phenotyping and geno-typing as 96 F6:7 recombinant inbred lines (RILs).
B35, Tx430, and the 96 F6:7 RILs were grown in field experi-ments under post-flowering drought stress conditions (stress) infour environments: Lubbock, Texas during 1993 (ENV1) and 1994(ENV2) and Halfway, Texas during 1993 (ENV3) and 1994(ENV4). Three irrigations were applied during the pre-floweringgrowth period to minimize the pre-flowering water deficit. Theexperiments were carried out in a randomized complete block de-sign with three replications. Each plot consisted of one row ofplants, 4.9 m long, with a 1.0 m row-spacing. Each replicationcontained random repetitions of the parents, B35 and Tx430, oncefor every 10 rows of RILs.
Plots were evaluated for the stay-green trait near the end of thelinear grain-fill period. At each environment, visual ratings wererecorded for expression of the stay-green trait (scores ranged from1 to 5 based on the degree of leaf and plant death; score 1 repre-sents no senescenced leaves and score 5 representing all senescedleaves). Chlorophyll index readings were taken with SPAD-502chlorophyll meter (Spectrum Technologies Inc), and were recordedat the same time only during the 1994 growing season to back-upthe visual ratings of stay-green. Maturity ratings were recorded asthe number of days from planting to 50% anthesis. Statisticalanalysis was performed using the Proc GLM (SAS Institute 1989)procedure to evaluate the parents and RILs for genetic variation instay-green and chlorophyll index assuming the fixed-eects model(Model I). Maturity ratings followed the normal distribution, whilelog transformation was done on the stay-green ratings to fit thenormality assumption. Broad-sense heritability (H) estimates werecalculated on a family-mean basis from pooled analysis as the ratioof genetic variance (r2g) to phenotypic variance (r2ph) (Fehr 1987).However, the random-eects model (Model II) was assumed forcalculation of heritability estimates.
Genomic DNA was isolated from the parental lines (B35 andTx430) and the 96 RILs (based on Saghai-Maroof et al. 1984).Southern blots were prepared by digesting 10 lg of DNA using fourrestriction endonucleases (EcoRI, EcoRV, BamHI, and HindIII;Promega, Madison, Wis.) and, following electrophoresis, transfer-ring the DNA to Hybond N+ membrane (Amersham Life Sci-ences, Arlington Heights, Ill.) as recommended by the membranemanufacturer.
The parental lines were screened for restriction fragment lengthpolymorphism (RFLP) using sorghum genomic clones (obtainedfrom Drs. G. Hart and A. Paterson; Texas A and M University),maize cDNA and genomic clones (Univ. of Missouri, Columbia),clones mapped on rice and wheat (Cornell University, N.Y.), andother cDNA clones available in our collection. Labeling of theprobes was done either by PCR-random primer labeling (BIOSTag-it-kit) or by oligolabeling (Feinberg and Vogelstein 1984). Theprobes that were polymorphic between the parental lines were usedfor hybridization with DNA from the 96 RILs and the parentallines. The RILs were scored for markers as A and B for presenceof the parental band of the female parent (B35) and male parent(Tx430), respectively, H for heterozygotes, C for non-femaleparent, D for non-male parent, and - for missing and non-pa-rental markers.
The RFLP linkage map was developed using MAPMAKER(Lander et al. 1987). A LOD score of 3.0 was used to establishlinkage between markers. The level of heterozygosity within each
line ranged from 0 to 24%, with an average of 11%, among co-dominant markers. Therefore the MAPMAKER option F2 in-tercross instead of RIL was used for developing the geneticlinkage map. However, the RIL option, which requires completehomozygosity, was tried and the resultant map order did notchange, although the map distances were aected because the he-terozygotes were treated as missing values. Genome compositionwas estimated based on marker genotypes and the map distancesbetween markers as described by Paterson et al. (1991).
Simple interval mapping (Paterson et al. 1988) was applied to de-termine the chromosomal locations of putative QTLs influencingstay-green and maturity traits, while the composite interval map-ping (Jansen 1993) method was applied to evaluate theQTL Environment interactions. Simple interval mapping wasdone using the MAPMAKER/QTL program (Lander and Botstein1989). The pooled means of the traits across four environmentswere used for mapping QTLs. A LOD threshold of 1.3 was initiallyused to identify putative QTLs (the LOD thresholds are oftenlowered to identify putative QTLs influencing complex traitswith large G E interactions; Stuber et al. 1992). The putativeQTLs were further evaluated in a step-wise manner using LODscore as selection criterion. All putative QTLs with a minimumLOD increment of 2.0 were considered as candidate QTLs.
Composite interval mapping was done using PLABQTL soft-ware (Utz and Melchinger 1996). The pooled means of traits wereused for mapping QTLs and the means from individual environ-ments were used to estimate the QTL Environment interaction.Initially, markers were selected as cofactors by stepwise regressionanalysis with a stringent F-enter and F-drop value of 5.0 each.These selected markers except those on the current chromosome were used as co-factors (cov /- command). A LOD threshold of2.0 was used to determine the presence of the candidate QTL. FinalQTL analysis was done by using the markers adjacent to the can-didate QTLs as cofactors with a LOD threshold of 2.0.
Out of the 142 RFLP markers used for mapping, 128markers were mapped to 14 linkage groups (LGs)spanning a total of 1602 centiMorgans (cM in the Ko-sambi function) (Fig. 1). The average distance betweenadjacent markers was 14.1 cM. Approximately 51% ofthe intervals between adjacent markers were smallerthan 20 cM and 26% were in the range from 20 to30 cM. Sixteen markers were scored as dominantmarkers, while 112 were scored as codominant markers.Average heterozygosity among markers within an indi-vidual ranged from 0 to 24%, with an average of 11%.Distorted segregation of homozygous alleles (expectedratio 1:1, excluding heterozygotes) occurred (P 0.05)at 17 RFLP loci.
On average, 43.5% of the genome was homozygousfor B35 alleles, 46.7% of the genome was homozygousfor Tx430 alleles, while 9.8% of the genome was het-erozygous (Fig. 2). The v2 test against the expected ratioof 48.4:3.1:48.4 for aa:ab:bb alleles for generation F6(since the RILs were grown from bulked seeds from F6lines) was significant (v2 14.8, k 2, P 0.0001).The distortion of the segregation ratio was solely due to
the increased proportion of heterozygotes compared tohomozygotes, as the proportion of homozygotes(43.5:46.7 for aa:bb) was within the expected limits for1:1 (v2 0.79, k 1, P 0.37). The segregation ratiowas equivalent to the expected ratio of 46.9:6.2:46.9 foraa:ab:bb alleles in the F5 generation (v2 2.26, k 2,P 0.32), suggesting that the development of RILsmay have been slowed down due to bulking of the seedsin the F3 generation and may not reflect strict single-seed descent breeding, which favors the taller and latermaturing heterozygous plants. This notion is strength-ened by the Poisson-type distribution of heterozygousgenome, where median and mode are more representa-tive measures of central tendency than mean (Fig. 2).
The stay-green ratings were significantly lower (greener)in B35 (pooled average of 1.8) than in Tx430 (pooledaverage of 3.4) at all four environments (Fig. 3). Thechlorophyll index values for B35 (48.5) were also sig-nificantly higher compared to Tx430 (28....